Indocyanine green-hyaluronic acid-polypeptide conjugate and its preparation method and application

Abstract

This invention provides an indocyanine green-hyaluronic acid-peptide conjugate, its preparation method, and its application, belonging to the field of biomedical technology. The structure of the conjugate is shown in formula (I). The conjugate provided in this application has good stability and biological activity, and can achieve a synergistic effect of tumor apoptosis and photothermal therapy, thus possessing good practical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an indocyanine green-hyaluronic acid-peptide conjugate, its preparation method, and its application. Background Art

[0002] Cancer has always been one of the leading killers threatening human life and a major challenge facing the global medical research field. Its high mortality rate, high incidence rate, and trend of affecting younger people make cancer a top priority for scientific research, thus making the search for cancer treatments an urgent priority.

[0003] Hyaluronic acid (HHA) can exert various structural functions in the extracellular matrix (ECM) through specific and non-specific interactions with a variety of functional proteins. It also plays a crucial role in cell-to-specific molecule / receptor communication transduction. For example, CD44, neuroglycans, glial hyaluronic acid-binding protein (GHAP), and lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) can all interact with HHA. Current research indicates that CD44 is highly expressed on the surface of various tumor cells, therefore HHA is considered a potential drug carrier for targeted therapy.

[0004] Anticancer peptides (ACPs) possess advantages such as broad-spectrum anticancer activity, low immunogenicity, rapid killing of cancer cells, certain selectivity, excellent tissue penetration, low likelihood of inducing drug resistance, and ease of synthesis. Furthermore, they are easily modified to enhance in vivo stability and biological activity, making them potential candidates for developing into next-generation antitumor drugs and ideal molecules for cancer treatment.

[0005] In recent decades, phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), has made significant progress in achieving anti-tumor effects. PTT is a method that uses photothermal agents to generate localized heat to destroy cancer cells. For example, gold nanoshells and indocyanine green (ICG) are common photothermal agents whose anti-tumor therapeutic effects have been evaluated in clinical trials. PDT utilizes photosensitizers to generate reactive oxygen species (ROS) to damage cancer cells, such as hematoporphyrin derivatives (HpD), and has been used clinically for over 40 years. For photothermal dynamic therapy of cancer, the development of high-quality therapeutic agents is crucial. Near-infrared (NIR) fluorescent dyes are widely popular due to their excellent properties, including stronger tissue penetration than visible light and immunity to tissue autofluorescence. Notably, indocyanine green (ICG) is the only NIR fluorescent dye approved for clinical use by the U.S. Food and Drug Administration (FDA). In recent years, ICG has been used as an excellent photothermal agent and photosensitizer in cancer phototherapy.

[0006] Existing technologies have provided several antitumor therapeutic agents containing photothermal agents. For example, Chinese invention patent CN118267343A provides a photothermal-photodynamic synergistic antibacterial freeze-dried microneedle containing hyaluronic acid-encapsulated ZIF-8 nanoparticles loaded with indocyanine green; Chinese invention patent CN117731775A provides a hyaluronic acid-modified drug-loaded nanocomposite containing hyaluronic acid and indocyanine green; and Chinese invention patent CN116712665A provides a photothermal-chemotherapy combined therapeutic agent, using microneedles to fill the cavities of the microneedles. However, the hyaluronic acid and photothermal agents in the above-mentioned therapeutic agents are mixed in an encapsulated or composite form, resulting in poor stability and easy detachment during application, especially after entering the human body circulation, which is not conducive to fully exerting the therapeutic effect of the photothermal agent. Chinese invention patent CN114349807A provides a sialic acid-linked indocyanine green (Sia-ICG) product, in which ICG is linked to sialic acid via an amide bond. However, as a monosaccharide, sialic acid is still metabolized relatively quickly in vivo, and the persistence of the photothermal agent's effect still needs to be improved.

[0007] Currently, there are no existing technologies that have explored how to use photothermal and photodynamic methods to synergistically treat tumors with anticancer peptides. Summary of the Invention

[0008] To address the above problems, this application proposes the following technical solution:

[0009] On the one hand, this application provides an indocyanine green-hyaluronic acid-peptide conjugate comprising the structural formula shown in formula (I):

[0010]

[0011] In formula (I), R1 is a polypeptide containing at least two amino acids, and R2 has the structure shown in formula (II):

[0012]

[0013] The value of n ranges from 1 to 25.

[0014] In one implementation, the value of n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.

[0015] In one embodiment, the value of n is preferably 5 to 25, and more preferably 8 to 12.

[0016] It is understood that the polypeptide described in this application is a compound formed by the dehydration condensation of at least two amino acid molecules linked by peptide bonds. In this application, it may be a small molecule peptide with known biological activity, such as antibacterial, antihypertensive, hypolipidemic, antithrombotic, anti-inflammatory, and antitumor effects.

[0017] In one embodiment, the imine bond adjacent to R1 originates from the N-terminus of the polypeptide. Specifically, the imine bond is formed by the reaction of the N-terminal amino group of the polypeptide with the aldehyde group of 4-carboxybenzaldehyde.

[0018] In one embodiment, the polypeptide is an anticancer peptide, such as TAT-KLA peptide.

[0019] In one embodiment, the polypeptide is a TAT-KLA peptide.

[0020] In one embodiment, the amino acid sequence of the TAT-KLA peptide from the N-terminus to the C-terminus is as follows:

[0021] YGRKKRRQRRR-GG-D(KLAKLAKKLAKLAK).

[0022] In one embodiment, the TAT-KLA peptide has a molecular weight of 3180.31 Da.

[0023] In the coupling shown in formula (I), the R2 group is derived from indocyanine green primary amine (ICG-NH2), with the molecular formula C 47 H 56 N4O4S, molecular weight 773.04.

[0024] In the conjugate shown in formula (I), R1 is a substituted TAT-KLA peptide, R2 is a substituted ICG-NH2, and hyaluronic acid serves as the molecular backbone carrier that simultaneously immobilizes both through chemical bonds. Specifically, the TAT-KLA peptide is linked to the carboxyl group of a portion of the disaccharide monomer of hyaluronic acid via a linker bridge formed by adipic acid dihydrazide (ADH) and 4-carboxybenzaldehyde (CBA). In the linker bridge, adipic acid dihydrazide is linked to HA via an amide bond, 4-carboxybenzaldehyde is linked to adipic acid dihydrazide via an amide bond, and the TAT-KLA peptide is linked to 4-carboxybenzaldehyde via an imine bond. The amino group of ICG-NH2 is linked to the carboxyl group of a portion of the disaccharide monomer of hyaluronic acid via an amide bond.

[0025] On the other hand, this application also provides a method for preparing the indocyanine green-hyaluronic acid-peptide conjugate, comprising:

[0026] S1. Hyaluronic acid and adipic acid dihydrazide are reacted to prepare the intermediate product HA-ADH;

[0027] S2. The HA-ADH obtained in step S1 is reacted with 4-carboxybenzaldehyde to obtain the intermediate product HA-ADH-CBA.

[0028] S3. React the polypeptide with the HA-ADH-CBA obtained in step S2 to obtain a hyaluronic acid-polypeptide conjugate.

[0029] S4. The remaining carboxyl groups in the monomer of the hyaluronic acid-peptide conjugate obtained in step S3 are subjected to an amidation reaction to obtain the indocyanine green-hyaluronic acid-peptide conjugate.

[0030] In one embodiment, the polypeptide is a TAT-KLA peptide.

[0031] In one embodiment, the hyaluronic acid has a molecular weight of 1000-20000 Da.

[0032] Preferably, the molecular weight of hyaluronic acid is 8000 Da.

[0033] In one embodiment, in S1, the molar ratio of hyaluronic acid to adipic dihydrazide is 1:(2-6), preferably 1:5.

[0034] In one embodiment, the molar ratio of HA-ADH and 4-carboxybenzaldehyde in S2 is 1:(1-3), preferably 1:1.5.

[0035] In one embodiment, in S3, the molar ratio of the polypeptide to HA-ADH-CBA is (0.5-5):(0.5-5), preferably 1:1.

[0036] In one embodiment, in step S4, the molar ratio of indocyanine primary amine to hyaluronic acid-peptide conjugate is (0.5-5):(0.5-5), preferably 1:1.

[0037] In one embodiment, a catalyst is added during the reactions S1, S2 and S4, the catalyst being 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS).

[0038] Preferably, the molar ratio of EDCI to NHS is 1:1.

[0039] In one embodiment, the reaction temperature of S1-S4 is 0-40°C, preferably 10-30°C, and the reaction time is 1-24h.

[0040] In one embodiment, a purification step may be performed after each step of the above method.

[0041] It is understood that those skilled in the art can use known methods in the art to select a suitable purification method for the conjugates of the present invention, such as dialysis and lyophilization. Specifically, dialysis can be performed using the dialysis bag method. The reaction product is poured into a pretreated dialysis bag, dialyzed with water or an ethanol / water mixture one to several times to remove impurities, filtered through a filter membrane, pre-frozen, and then lyophilized in a freeze dryer.

[0042] Preferably, the dialysis uses a dialysis bag with a molecular weight cutoff of 1000-3500.

[0043] After purification, IHTK is subjected to ultrasonic treatment, which allows it to form a well-dispersed, uniformly sized spherical structure in a dissolution medium.

[0044] Optionally, the method for preparing the ICG-HA-TAT-KLA conjugate described above specifically includes:

[0045] Preparation of HA-ADH conjugate: HA is dissolved in water, EDCI and NHS are added as catalysts to activate the carboxyl group in HA, and then a completely dissolved ADH aqueous solution is added to continue the reaction. After the reaction is completed, the reaction product is purified to obtain the HA-ADH conjugate.

[0046] Preparation of HA-ADH-CBA conjugate: CBA was dissolved in dimethyl sulfoxide, and EDCI and NHS were added as catalysts to activate the carboxyl group in CBA; HA-ADH was dissolved in water, and the above CBA solution was slowly added to continue the reaction. After the reaction was completed, the reaction product was purified to obtain the HA-ADH-CBA conjugate.

[0047] Preparation of HTK conjugates: Dissolve HA-ADH-CBA in a mixture of buffer (pH=7.4) and dimethyl sulfoxide, add TAT-KLA, and react overnight at room temperature. After the reaction is complete, the reaction product is purified to obtain the HTK conjugates.

[0048] Preparation of IHTK conjugates: HTK is dissolved in water, and EDCI and NHS are added as catalysts to activate the carboxyl groups in HTK; ICG-Amine is dissolved in dimethyl sulfoxide and slowly added to the completely dissolved HTK solution. After the reaction is completed, the reaction product is purified to obtain the IHTK conjugates; after ultrasonic treatment, well-dispersed and uniformly sized spherical structures can be formed in the dissolution medium.

[0049] In the preparation of HA-ADH conjugate, the activation time was controlled at 20 min; the above adipic acid dihydrazide solution was added to continue the reaction, and the reaction time was controlled at 6 h; the purification steps of the reaction product included dialysis and lyophilization.

[0050] In the preparation of HA-ADH-CBA conjugate, the activation time was controlled at 20 min; the above-mentioned 4-carboxybenzaldehyde solution was slowly added to continue the reaction, and the reaction time was controlled at 6 h; the purification steps of the reaction product included dialysis and lyophilization.

[0051] In the preparation of HTK conjugates, the buffer solution is citrate-disodium hydrogen phosphate buffer or disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. After adding TAT-KLA, the reaction time is controlled at 12h. The purification steps of the reaction product include dialysis and drying.

[0052] During the preparation of the IHTK conjugate, the activation time was controlled at 20 min; the above ICG-Amine solution was slowly added to continue the reaction, and the reaction time was controlled at 6 h; the purification steps of the reaction product included dialysis and lyophilization; the ultrasonic power was 40 W and the time was 5 min.

[0053] On the other hand, this application also provides an antitumor pharmaceutical formulation comprising the indocyanine green-hyaluronic acid-peptide conjugate and at least one pharmaceutically acceptable excipient.

[0054] On the other hand, this application also provides an antitumor pharmaceutical composition comprising the indocyanine green-hyaluronic acid-peptide conjugate described above.

[0055] It is understood that the pharmaceutical compositions or pharmaceutical preparations of the present invention can be administered intravenously, via the gastrointestinal tract, or in other pharmaceutically acceptable forms, including but not limited to injections, oral preparations, injectable implants, emulsions, liposomes, microcapsules, microspheres, nanoparticles, etc.

[0056] It is understood that commonly used pharmaceutically acceptable excipients are excipients, such as solvents, binders, fillers, wetting agents, etc., which can be selectively added when preparing a specific dosage form. If necessary, other excipients such as flavoring agents, coloring agents, stabilizers, and lubricants can also be added. Those skilled in the art can use techniques known in the art to formulate the conjugates of the present invention into pharmaceutical compositions or pharmaceutical preparations. For example, pharmaceutical preparations can be prepared according to the Modern Pharmaceutical Preparations Series edited by Shenyang Pharmaceutical University. Furthermore, in addition to those mentioned in this invention, suitable pharmaceutical excipients can also be other types known in the art, such as those described in the *Handbook of Parmaceutical Excipients*, edited by Paul J. Sheskey et al., which is currently in its eighth edition, with the first edition published in 1986 and the eighth edition published in 2017.

[0057] It is understood that the drug carrier can be a pharmaceutically acceptable solvent, suspension, or carrier for delivering the active pharmaceutical ingredient into animals or humans. The carrier can be liquid or solid and is selected according to the planned route of administration. Proteins and liposomes are also drug carriers.

[0058] On the other hand, this application also provides the indocyanine green-hyaluronic acid-peptide conjugate, or the indocyanine green-hyaluronic acid-peptide conjugate prepared by the method, or the pharmaceutical formulation, or the use of the pharmaceutical composition in the preparation of antitumor drugs.

[0059] In one embodiment, the tumor includes melanoma, breast tumor, lung tumor, colon tumor, ovarian tumor, and kidney tumor.

[0060] In one embodiment, the antitumor drug is applied by irradiating the tumor site with a near-infrared light source.

[0061] On the other hand, this application also provides a system for tumor treatment, the system comprising:

[0062] a) the indocyanine green-hyaluronic acid-peptide conjugate described above, or the indocyanine green-hyaluronic acid-peptide conjugate prepared by the method described above, or the pharmaceutical formulation described above, or the pharmaceutical composition described above; and

[0063] b) An illumination device equipped with a near-infrared light source.

[0064] The light source emitted by the illumination device is a near-infrared light source, specifically, the wavelength of the near-infrared light source can be 808nm. Furthermore, experimental verification has shown that the conjugate provided in this application effectively inhibits tumor growth under 808nm laser irradiation, resulting in tumor ablation and achieving good photothermal and photodynamic therapy effects.

[0065] This application has at least the following technical advantages or effects:

[0066] 1. This invention uses a derivative obtained by linking adipic acid dihydrazide and 4-carboxybenzaldehyde as an acid-sensitive linker to link the crude apoptosis peptide TAT-KLA synthesized in a chemical solid phase to a hydrophilic hyaluronic acid molecular backbone, and indocyanine green and HA are linked by an amide bond, thereby successfully preparing the ICG-HA-TAT-KLA conjugate. In this conjugate, the hyaluronic acid carrier has a good targeting effect on tumor cells, and can rapidly and effectively deliver anticancer peptides and ICG to the tumor cell site. TAT in the TAT-KLA peptide is a membrane-penetrating peptide with extremely strong cell membrane penetration ability, and KLA is a safe pro-apoptotic peptide. The ADH-CBA linker can release the TAT-KLA peptide into a free state under the acidic conditions of the tumor microenvironment, thereby significantly improving the therapeutic efficiency of TAT-KLA peptide at the tumor cell site. At the same time, after ICG reaches the tumor site, it can exert photothermal and photodynamic effects through near-infrared light irradiation. On the one hand, it can promote tumor cell apoptosis, and on the other hand, it can also help promote the release of TAT-KLA peptide, significantly increasing its release rate in the tumor micro-acidic environment, and further playing a synergistic role in tumor treatment.

[0067] 2. The IHTK conjugate prepared in this application immobilizes the anticancer peptides TAT-KLA and ICG on a hyaluronic acid molecular carrier via chemical bonds, exhibiting better stability after entering the human body compared to encapsulation or filling methods. Simultaneously, compared to monosaccharides or other molecular carriers, HA in polysaccharide form can self-assemble into nanoparticles. Nanoparticle drug carriers can increase drug concentration at the target site, thereby improving drug utilization and efficacy, and reducing adverse drug reactions. Furthermore, the polysaccharide structure of HA is metabolized more slowly in vivo, and its more complex structure protects the internal chemical bonds from enzymatic degradation to some extent, further enhancing its stability and persistence in vivo, and increasing the content of anticancer peptides and ICG at the tumor site.

[0068] 3. The method for synthesizing IHTK conjugates provided in this application can successfully produce end products with physicochemical properties more suitable for practical applications. Specifically, the introduction of an appropriate amount of ICG can fully utilize the photothermal and photodynamic therapeutic effects while avoiding physiological discomfort and damage to normal cells caused by excessively high local temperatures, and even affecting the efficacy of anticancer peptides. Furthermore, an appropriate molecular weight of hyaluronic acid carrier not only improves product yield, but the resulting conjugate also has excellent water solubility, enabling it to be formulated into various dosage forms such as intravenous injections in practical applications or production. Moreover, it can be rapidly and effectively absorbed after entering the human body, showing better application prospects. Attached Figure Description

[0069] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0070] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0071] Figure 1 This is a synthetic route diagram of the IHTK conjugate in Example 1;

[0072] Figure 2 The 1H NMR spectrum of the HTK conjugate in Example 1;

[0073] Figure 3 The near-infrared absorption spectrum of the IHTK conjugate in Example 1 is shown below.

[0074] Figure 4 The image shows the particle size distribution of the IHTK coupling material in Example 1.

[0075] Figure 5 The results of the environmentally responsive release behavior evaluation of the IHTK conjugate in Test Example 1;

[0076] Figure 6 The results of testing the apoptotic effects of TAT-KLA, HTK conjugates and IHTK conjugates in Example 2 on melanoma cells;

[0077] Figure 7 The results of HE staining of melanoma were tested using TAT-KLA, HTK conjugates and IHTK conjugates in Example 3. Detailed Implementation

[0078] To more clearly illustrate the overall concept of this application, a detailed description is provided below by way of embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of this application. However, it will be apparent to those skilled in the art that this application can be implemented without one or more of these details. In other instances, to avoid confusion with this application, some technical features well-known in the art have not been described.

[0079] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the experimental methods used in the following preparation examples and embodiments are conventional methods, and the reagents used in the preparation examples and embodiments are reagents supplied by conventional reagent manufacturers unless otherwise specified.

[0080] Information on the experimental reagents and instruments used in the following examples is shown in Table 1:

[0081] Table 1. Reagent and Instrument Information

[0082]

[0083] In the following embodiments, the structural formula of the HA-TAT-KLA conjugate is HA~ADH-CBA~TAT-KLA, abbreviated as HTK. In the embodiments of the present invention, the HA-TAT-KLA conjugate described in the present invention can be represented in the form of the structural formula or in the form of HTK.

[0084] In the following embodiments, the structural formula of the ICG-HA-TAT-KLA conjugate is ICG~HA~ADH-CBA~TAT-KLA, abbreviated as IHTK. In the embodiments of the present invention, the ICG-HA-TAT-KLA conjugate of the present invention can be represented in the form of the structural formula or in the form of IHTK.

[0085] In the following examples, the amino acid sequence of the TAT-KLA peptide is as follows:

[0086] Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Gly-Gly-Lys-Leu-Ala-Lys-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Leu-Ala-Lys,

[0087] The abbreviation is: YGRKKRRQRRR-GG-D(KLAKLAKKLAKLAK),

[0088] Its molecular weight is 3180.31 Da.

[0089] Example 1: Preparation of ICG-HA-TAT-KLA conjugate (ICG~HA~ADH-CBA~TAT-KLA, IHTK)

[0090] This embodiment provides a method for preparing the ICG-HA-TAT-KLA conjugate, and the specific preparation steps are as follows:

[0091] (1) Dissolve 0.5 mmol of HA (molecular weight 8000 Da, n≈10) in deionized water to obtain an HA solution. Add 1.2 mmol of EDCI and 1.2 mmol of NHS as catalysts in a 1:1 ratio and react for 20 min. Separately, weigh 2.5 mmol of ADH, dissolve it in deionized water, and add it dropwise to the catalyzed HA solution. React at room temperature for 6 h. Then, place the solution in a dialysis bag (molecular weight cutoff 1000) and dialyze it three times with deionized water to remove unreacted impurities. Filter through a 0.22 μm filter membrane, pre-freeze, and freeze-dry in a freeze dryer to obtain the HA-ADH conjugate.

[0092] (2) Dissolve 0.5 mmol of HA-ADH in deionized water to obtain an HA-ADH solution. Separately, weigh 0.75 mmol of CBA and dissolve it in dimethyl sulfoxide. Add EDCI and NHS as catalysts in a 1:1 ratio and react for 20 min. Slowly add the solution dropwise to the completely dissolved HA-ADH and react at room temperature for 6 h. Then, place the solution in a dialysis bag (molecular weight cutoff 1000) and dialyze it three times with an ethanol / water (V:V = 1:4) mixture to remove unreacted impurities. Filter through a 0.22 μm filter membrane, pre-freeze, and freeze-dry to obtain the HA-ADH-CBA conjugate.

[0093] (3) Dissolve 0.05 mmol of HA-ADH-CBA conjugate in a mixture of buffer (pH=7.4) and DMSO (V:V=1:1), add 0.05 mmol of TAT-KLA, and react at room temperature for 12 h. Then, place the mixture in a dialysis bag (molecular weight cutoff 3000) and dialyze three times with pH 7.4 buffer to remove unreacted impurities. After filtration through a 0.22 μm filter membrane, pre-freeze the mixture and freeze-dry it in a freeze dryer to obtain the HTK conjugate.

[0094] (4) Dissolve 0.05 mmol of HTK in buffer solution (pH = 7.4) to obtain an HTK solution. Add EDCI and NHS as catalysts in a 1:1 ratio and react for 20 min. Separately, weigh 0.05 mmol of ICG-Amine and dissolve it in dimethyl sulfoxide. Slowly add this solution to the catalyzed HTK solution and react at room temperature for 6 h. Then, place the solution in a dialysis bag (molecular weight cutoff 1000) and dialyze three times with an ethanol / water (V:V = 1:4) mixture to remove unreacted impurities. Filter through a 0.22 μm filter membrane, pre-freeze, and freeze-dry to obtain the IHTK conjugate. The calculated yield is approximately 15%.

[0095] When IHTK conjugates are treated with ultrasound, they can form well-dispersed, uniformly sized spherical structures in a dissolution medium.

[0096] The synthetic route of the above embodiments is as follows: Figure 1 As shown.

[0097] The chemical structure of the intermediate HTK was identified by 1H NMR, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the intermediate product prepared in this embodiment is confirmed to be an HTK conjugate.

[0098] The structure of the IHTK conjugate was identified using near-infrared spectroscopy absorption, and the results are as follows: Figure 3 As shown. By Figure 3 The results show that the final product obtained in this embodiment is confirmed to be an IHTK conjugate.

[0099] The morphology of the IHTK conjugate was observed using scanning electron microscopy, and the results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the IHTK conjugate prepared in this embodiment has a spherical structure at the microscopic level.

[0100] Optimization of the synthesis process:

[0101] The embodiments of this application also optimize the synthesis method of IHTK conjugates. Specifically, the method described in Example 1 is used to prepare IHTK conjugates with hyaluronic acid of different average molecular weights, namely 4000Da (n≈5), 8000Da (n≈10), 16000Da (n≈20), and 32000Da (n≈40).

[0102] However, experiments showed that the yields of the aforementioned hyaluronic acid raw materials in IHTK conjugates were approximately 4%, 15%, 13%, and 10%, respectively. This indicates that the IHTK conjugates prepared using the method provided in this application can achieve yields of over 15%. When using hyaluronic acid with a lower molecular weight as the molecular backbone, the fewer molecular chains result in insufficient collisions between the carboxyl groups and ICG, leading to a lower yield. Conversely, when using hyaluronic acid with an excessively high molecular weight, its easily cross-linked structure in space also reduces the synthesis efficiency of ICG. Furthermore, the water solubility of the final product obtained from hyaluronic acid with a molecular weight of 32000 Da is reduced, making spectral characterization difficult and unsuitable for practical applications such as intravenous injections, as its absorption after entering the human body is also impaired. Therefore, considering both the product preparation yield and its physicochemical properties, the preferred molecular weight of the raw hyaluronic acid is 5000–20000 Da, i.e., n≤25.

[0103] Test Example 1

[0104] This test case evaluates the environmentally responsive release behavior of the IHTK conjugate prepared in Example 1. The experimental procedure is as follows:

[0105] (1) Experimental drug: IHTK conjugate prepared in Example 1

[0106] (2) Experimental Methods: To verify whether the synthesized IHTK is responsive in an acidic environment, the cumulative release of TAT-KLA peptide was detected by fluorescence spectrophotometry. 1 mL of TAT-KLA peptide at a concentration of 100 μg / mL IHTK was placed in 20 mL buffer solutions containing different pH values ​​(7.4, 6.8, 5.0) for in vitro simulated acid sensitivity experiments. Each solution was incubated in a shaker at 37°C. Samples of the release medium were taken at different time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72 h). Each time, 0.5 mL of the sample was taken, and an equal amount of fresh release medium was added. The TAT-KLA content was determined using fluorescence spectrophotometry with a multi-functional microplate reader, and time-release curves were plotted. The evaluation results of the environmental responsiveness release behavior of the conjugate are shown in [the table below]. Figure 5 And Table 2.

[0107] Table 2

[0108]

[0109]

[0110] From Table 2 and Figure 5 It was found that almost no TAT-KLA peptide was released in a neutral environment at pH 7.4. The cumulative release rate increased with increasing acidity, with release rates reaching 69% and 86% after 72 hours at pH 6.8 and pH 5.0, respectively. This is significantly higher than the release rates of the hyaluronic acid-peptide conjugate (disclosed in patent CN113648427B) (approximately 45% at pH 6.8 and approximately 80% at pH 5.0 after 72 hours). This indicates that the IHTK conjugate has good acid-sensitive responsiveness, enabling rapid release of TAT-KLA peptide in the acidic tumor microenvironment, thus exerting a better therapeutic effect. Furthermore, the introduction of ICG helps to enhance the release of TAT-KLA peptide in an acidic environment, thereby further improving the therapeutic efficacy of the anticancer peptide TAT-KLA.

[0111] Test Example 2

[0112] This test case compares the apoptotic effects of TAT-KLA peptide, HTK conjugate, and IHTK conjugate on B16 melanoma cells. The experimental procedure is as follows:

[0113] (1) Experimental drugs: The blank group PBS solution and the control group were TAT-KLA peptide (TK group), HTK conjugate (HTK group), IHTK conjugate without laser irradiation (IHTK-L group), and IHTK conjugate with laser irradiation (IHTK+L group), respectively.

[0114] (2) Experimental method: The toxic side effects of different concentration ranges of TAT-KLA, HTK and IHTK on B16 cells were measured and analyzed using CCK-8.

[0115] Detailed operation process:

[0116] 1×10 B16 melanoma cells 4 The contents were added to 96-well plates and incubated overnight. Different concentrations (containing 2 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml of TAT-KLA peptide, respectively) of TAT-KLA, HTK, and IHTK were added and incubated for 24 hours. PBS served as a control group. IHTK was divided into a laser-irradiated group (IHTK+L) and a non-laser-irradiated group (IHTK-L). The IHTK+L group was irradiated with an 808 nm laser for 10 min (1 W / cm²). 2 After stabilizing in a sterile incubator containing 5% carbon dioxide for 6 hours, 10 μL of CCK-8 solution was added to each sample well. The 96-well plate was then incubated at 37°C for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the viability of B16 melanoma cells was calculated. The results of the CCK-8 apoptosis-inducing assay are shown below. Figure 6 .

[0117] Depend on Figure 6 It is known that TAT-KLA, HTK and IHTK can all promote tumor apoptosis. The apoptosis-promoting ability of HTK and IHTK is significantly enhanced. Furthermore, after near-infrared laser irradiation, IHTK exerts photothermal and photodynamic therapeutic effects, further enhancing its apoptosis-promoting ability.

[0118] Test Example 3

[0119] This test case examines the tumor-inhibiting effects of TAT-KLA peptide, HTK conjugate, and IHTK conjugate on tumor-bearing mice. The experimental procedure is as follows:

[0120] (1) Experimental drugs: The blank group PBS solution and the control group were TAT-KLA peptide (TK group), HTK conjugate (HTK group), IHTK conjugate without laser irradiation (IHTK-L group), and IHTK conjugate with laser irradiation (IHTK+L group), respectively.

[0121] (2) 2×10 5Prepared B16 cells were injected subcutaneously into BALB / c mice for tumor implantation. The tumors were allowed to grow to approximately 100 mm in size. 3 Following this, 100 μL of PBS solution was injected via the tail vein as a blank control group. The control group received 100 μL of solutions containing 150 nmol of TK, HTK, and IHTK. Two hours after injection, the tumors in the IHTK+L group were irradiated with an 808 nm laser for 10 minutes (1 W / cm²). 2 The mice were treated under the same conditions five times. At 24 days, the mice were euthanized, and the tumors were dissected for photography and data analysis. Tumor HE staining results are shown below. Figure 7 .

[0122] Depend on Figure 7 It can be seen that HTK and IHTK have a significant inhibitory effect on tumors, and the IHTK laser group has the most significant tumor-suppressing effect.

[0123] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An indocyanine green-hyaluronic acid-peptide conjugate, characterized in that, It contains the structural formula shown in equation (I): In formula (I), R1 is a TAT-KLA peptide, and R2 has the structure shown in formula (II): The value of n is 10.

2. The method for preparing the indocyanine green-hyaluronic acid-peptide conjugate as described in claim 1, characterized in that, include: S1. Hyaluronic acid and adipic acid dihydrazide are reacted to obtain the intermediate product HA-ADH, wherein the molecular weight of the hyaluronic acid is 8000 Da; S2. The HA-ADH obtained in step S1 is reacted with 4-carboxybenzaldehyde to obtain the intermediate product HA-ADH-CBA. S3. React the TAT-KLA peptide with the HA-ADH-CBA obtained in step S2 to obtain the hyaluronic acid-peptide conjugate. S4. The remaining carboxyl groups in the monomer of the hyaluronic acid-peptide conjugate obtained in step S3 are subjected to an amidation reaction to obtain the indocyanine green-hyaluronic acid-peptide conjugate.

3. The method according to claim 2, characterized in that, In S1, the molar ratio of hyaluronic acid to adipic dihydrazide is 1:(2-6); And / or, in S2, the molar ratio of HA-ADH to 4-carboxybenzaldehyde is 1:(1-3); And / or, in S3, the molar ratio of TAT-KLA peptide to HA-ADH-CBA is (0.5-5):(0.5-5); And / or, in S4, the molar ratio of indocyanine to hyaluronic acid-peptide conjugate is (0.5-5):(0.5-5); And / or, a catalyst is added during the reactions of S1, S2 and S4, the catalyst including EDCI and NHS; And / or, the reaction temperature of S1-S4 is 0-40℃ and the reaction time is 1-24h.

4. A pharmaceutical composition, characterized in that, It contains the indocyanine green-hyaluronic acid-peptide conjugate as described in claim 1.

5. A pharmaceutical preparation, characterized in that, It comprises the indocyanine green-hyaluronic acid-peptide conjugate as described in claim 1 and at least one pharmaceutically acceptable excipient.

6. The use of the indocyanine green-hyaluronic acid-peptide conjugate as described in claim 1, or the indocyanine green-hyaluronic acid-peptide conjugate prepared by the method as described in claim 2 or 3, or the pharmaceutical composition as described in claim 4, or the pharmaceutical formulation as described in claim 5, in the preparation of an anti-melanoma drug.

7. The application according to claim 6, characterized in that, When the anti-melanoma drug is applied, a near-infrared light source is used to irradiate the tumor site.

8. A system for treating melanoma, characterized in that, The system includes: a) the indocyanine green-hyaluronic acid-peptide conjugate as claimed in claim 1, or the indocyanine green-hyaluronic acid-peptide conjugate prepared by the method as claimed in claim 2 or 3, or the pharmaceutical composition as claimed in claim 4, or the pharmaceutical formulation as claimed in claim 5; and b) An illumination device equipped with a near-infrared light source.

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